DAVID MEALING

Abstract

A study has been made of the effects of various treatments on the phagocytosis of 14C-labelled E.coli by amoebae of Dictyostelium discoideum. An assay was also developed for the adhesion of amoebae to glass and the effects of a number of inhibitors on this process have been investigated. The phagocytosis of bacteria was inhibited by chelating agents at millimolar concentrations. The effect of chelators was not apparent in the presence of added divalent cations. However, only a small reduction in cell-glass adhesion was seen with EDTA concentrations that caused large reductions in phagocytosis. The calcium ionophore, A23187 abolished phagocytosis at 40 ug/ml. Pretreatment of amoebae with lanthanum ions completely inhibited both phagocytosis and cell-glass adhesion at low concentrations. Both phagocytosis and adhesion to glass are also strongly inhibited by calmodulin antagonists. Neither cytochalasin B or colchicine affected phagocytosis. Concanavalin A strongly inhibited phagocytosis presumably due to a direct interaction with cell surface glycoproteins, since the effect did not occur in the presence of alpha-methyl mannoside. Both phagocytosis and adhesion to glass were greatly reduced on treatment of the amoebae with tunicamycin, again suggesting glycoprotein involvement. Pretreatment of amoebae for 30 min with 1 mg/ml trypsin or pronase had no effect on phagocytosis, although pretreatment with papain at the same concentration caused some reduction. However, phagocytosis became pronase sensitive on exposure to tunicamycin. Beta-glucosidase also caused a small but consistent reduction in phagocytosis and cell-glass adhesion. Phagosomes were isolated from amoebae by two procedures. In the first, cells were allowed to phagocytose 1 um diameter polystyrene beads. The endocytosed beads were then isolated by flotation on a discontinuous sucrose density gradient. In a second procedure, devised during the course of this work, an attempt was made to isolate phagosomes from ingested glutaraldehyde-fixed E.coli. Analysis of these preparations by SDS-polyacrylamide gel electrophoresis showed a number of differences between them. A comparison of these preparations with "bulk" plasma membrane revealed a considerable similarity of the polypeptide profile with that isolated using fixed E. coli.

Document Type

Thesis

Publication Date

1987

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